Pharmacological agents are being developed to modulate phosphotyrosyl (pTyr) dependent cell signalling. Emphasis is on inhibitors of pTyr-dependent binding interactions, which are mediated by src homology 2 (SH2) domains and on protein- tyrosine phosphatase (PTP) inhibitors. In the SH2 domain area, development of cell-permeable growth factor receptor-bound protein 2 (Grb2) antagonists is being undertaken as potential new therapeutics for a variety of cancers including erbB-2 and Met dependent cancers. Previously we had prepared a series of pTyr-mimicking amino acid analogues that were stable toward enzymatic degradation. These were incorporated into tripeptide platforms and shown to have high Grb2-SH2 domain-binding affinity. During the reporting period novel macrocycles were prepared that represented conformationally constrained tetrapeptide-mimicking variants of our earlier tripeptide inhibitors. In extracellular Grb2-SH2 domain binding assays, these new macrocyclic compounds displayed significantly enhanced potency. For certain analogues, binding constants in the low picomolar range were observed. These analogues represent the most potent synthetic antagonists reported against any SH2 domain. Importantly, in whole cell assays these macrocyclic ligands maintained superior potency, with in vivo inhibition constants against Grb2 SH2 domain binding being in the low nanomolar range. In culture against breast cancer cells that are mitogenically-driven through erB-2 dependent pathways that utilize Grb2, these macrocycles displayed cytostatic effects at submicromolar concentrations. The same compounds were non-toxic against similar cells that are not dependent on Grb2 pathways for survival. In a parallel collaboration effects were studied against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb2-dependent signaling pathways. In cellular studies, these agents inhibit hepatocyte growth factor (HGF)-induced cell migration in Met-containing fibroblasts at nanomolar concentrations and inhibit tubule formation potentially involved in angiogenesis. Work continues on the design, synthesis and biological evaluation of new macrocyclic Grb2 SH2 domain-signaling inhibitors. In the phosphatase area, a structure-based approach toward PTP inhibitor design is being pursued. Using as a display platform, a tripeptide sequence derived from an epidermal growth factor receptor (EGFr) autophosphorylation site, we examined a panel of synthetic pTyr mimetics for inhibitory potencies against YopH, which is a pathogenic PTP component of Yersinia pestis, the causative agent of plague. Certain of these tripeptides exhibited binding constants in the single-digit micromolar range. Work is ongoing to optimize these tripeptide leads as potential therapeutics for the treatment of plague. Such therapeutics may have value against the use of Yersinia pestis as a bioterrorism agent.